US8962569B2 - Compositions comprising spp24 peptide fragments - Google Patents

Compositions comprising spp24 peptide fragments Download PDF

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US8962569B2
US8962569B2 US13/699,288 US201113699288A US8962569B2 US 8962569 B2 US8962569 B2 US 8962569B2 US 201113699288 A US201113699288 A US 201113699288A US 8962569 B2 US8962569 B2 US 8962569B2
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spp24
tumor
protein
cells
bmp
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US20130143811A1 (en
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Samuel S. Murray
Elsa J. Murray
Jeffrey C. Wang
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US Department of Veterans Affairs
University of California Berkeley
University of California San Diego UCSD
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US Department of Veterans Affairs
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Assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA reassignment THE REGENTS OF THE UNIVERSITY OF CALIFORNIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WANG, JEFFREY C.
Assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA reassignment THE REGENTS OF THE UNIVERSITY OF CALIFORNIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WANG, JEFFREY C.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to a tumor suppression composition and methods of making and using the same.
  • Secreted phosphoprotein-24 kDa (UniProtKB, accession number: Q27967) is a glycoprotein that was first cloned from bovine bone matrix and subsequently found in the periosteum and liver, but not in the heart, lung, kidney, or spleen (Hu, B., et al., J. Biol. Chem., 270, 431-436 (1995); Erratum (corrected accession number): J. Biol. Chem., 270, 10359). It was later cloned in the mouse kidney (Okazaki, Y., et al., Nature, 420, 563-573 (2002)) and uterus (Strausberg, R., et al.
  • Bovine spp24 is transcribed as a 203 amino acid residue protein. The first 23 residues constitute a signal peptide, which is cleaved to produce a mature 180 amino acid residue protein with a calculated mass of 20.5 kDa and a pI of 7.9 prior to modification.
  • the N-terminal 107 amino acid residues of the mature protein constitute a cystatin or cysteine protease inhibitor domain, initially suggesting that one physiological role of spp24 might be to inhibit the cysteine proteases, such as cathepsin K, essential for bone resorption (Hu, 1995).
  • Small amounts of spp24 were later found in the fetuin-mineral complex (FMC), a high molecular mass complex of calcium phosphate mineral, fetuin, and matrix Gla protein (MGP) initially discovered in the serum of etidronate-treated rats (Price, P. A., et al., J. Biol. Chem., 278, 22153-22160 (2003)).
  • FMC fetuin-mineral complex
  • MGP matrix Gla protein
  • spp24 like fetuin and MGP, may inhibit calcification (Price, 2003).
  • spp24 which contains a heavily phosphorylated serine-rich domain, may accumulate in bone and mineral complexes due to nonspecific ionic interactions with calcium (Hu, 1995).
  • BMP is known to be an osteoinductive protein and can plan a role in tumor growth
  • functions and properties of spp24 with respect to tumor growth are otherwise largely unknown.
  • development of tumor such as cancer, particularly metastastic cancer, continues to pose major public health concerns, particularly among the aging population.
  • the gene (which is also known as SP2 or secreted phosphoprotein-2, vs. SP1 or osteopontin) is hereafter referred to as SPP24, while the protein containing 180 amino acid residues will be referred to as spp24 as noted in the original descriptions (Hu, 1995, Price, 2003), with amino acid residue numbers added as necessary for clarity.
  • composition comprises a bioactive agent in an effective amount that sequesters:
  • BMP bone morphogenetic protein
  • TGF- ⁇ transforming growth factor-beta family of cytokines
  • composition is effective for a disorder in a mammalian subject.
  • the bioactive agent comprises secreted phosphorprotein 24 kD (spp24) (SEQ ID NO:1) ( FIG. 2 ) or a fragment thereof, wherein the spp24 is in an effective amount for suppressing or delaying tumor growth.
  • the spp24 is capable of sequestering one of bone morphogenetic protein-2 (BMP-2) and morphogenetic protein-7 (BMP-7) and as least one transforming growth factor-beta (TGF- ⁇ ) family of cytokines so as to suppress or slow tumor growth in a mammalian subject.
  • BMP-2 bone morphogenetic protein-2
  • BMP-7 morphogenetic protein-7
  • TGF- ⁇ transforming growth factor-beta family of cytokines
  • the tumor can be any tumor.
  • the tumor is a metastatic cancer or a primary cancer.
  • the cancer can be metastatic or primary tumors selected from a tumor in bone, a lung tumor, a liver tumor, a brain tumor, a spinal tumor, a breast cancer, a prostate cancer, or any tumor type or individual tumor the growth of which is enhanced by growth factors of the TGF-beta family (e.g., a tumor that manifests with osteoblastic and/or bone metastases).
  • the fragment of spp24 has a molecular weight from about 14 kD to about 20 kD.
  • the composition can be formulated in a formulation suitable for systemic or local delivery.
  • the formulation is a systemic delivery formulation.
  • the formulation is a local delivery formulation.
  • local delivery formulation include, e.g., formulation for local injection or injection into a tumor, and a sustained release formulation.
  • systemic delivery formulation can be, e.g., formulation for intravenous injection or subcutaneous injection.
  • sustained release formulation can comprise, e.g., an implant or a patch to be administered at a site needing treatment.
  • the composition can be a local delivery formulation comprising a patch suitable for delivery to the site of tumor.
  • composition of the various embodiments above can further comprise a pharmaceutically acceptable carrier.
  • a method of treating or ameliorating a tumor comprising administering to a patient a composition comprising spp24 or a fragment thereof, and wherein the spp24 is in an effective amount for suppressing or delaying tumor growth.
  • the spp24 is capable of sequestering one of bone morphogenetic protein-2 (BMP-2) and morphogenetic protein-7 (BMP-7) and possibly as least one transforming growth factor-beta (TGF- ⁇ ) family of cytokines so as to suppress or slow tumor growth in a mammalian subject.
  • the fragment of spp24 has a molecular weight from about 14 kD to about 20 kD.
  • the spp24 fragment can have amino acids 80-129.
  • the composition can be formulated in a formulation suitable for systemic or local delivery.
  • the formulation is a systemic delivery formulation.
  • the formulation is a local delivery formulation.
  • local delivery formulation include, e.g., formulation for local injection or injection into a tumor, and a sustained release formulation.
  • systemic delivery formulation can be, e.g., formulation for intravenous injection or subcutaneous injection.
  • sustained release formulation can comprise, e.g., an implant or a patch to be administered at a site needing treatment.
  • the composition can be a local delivery formulation comprising a patch suitable for delivery to the site of tumor.
  • composition of the various embodiments above can further comprise a pharmaceutically acceptable carrier.
  • a method of making a composition comprises forming a composition of the various embodiments described above and below.
  • a method of producing recombinant spp24 or a fragment thereof comprises expressing a gene comprises a nucleotide sequence encoding spp24 (SPP24) in an expression system.
  • SPP24 comprises a nucleotide sequence selected from SEQ ID NO:2 ( FIG. 2 ).
  • the expression system comprises a mammalian cell, a plant cell, yeast, bacteria or is a cell-free expression system.
  • the expression system comprises E. coli.
  • FIG. 1 shows the structure of SPP24/pET20b.
  • FIG. 2 shows the sequence of the SPP24 insert (SEQ ID NOS: 1, 2, and 33) in SPP24/pET20b.
  • FIG. 3 shows the chromatogram showing fractionation of unbound inclusion body protein (Fractions 6-9) and His 6 -tagged spp24 (Fractions 19-23) by FF IMAC chromatography.
  • FIG. 4 shows Coomassie-blue stained 4% to 20% polyacrylamide gradient gels and Western blots showing proteins in freshly-prepared IMAC column fractions.
  • Lane 1 Molecular weight standards.
  • Lane 2 Unbound proteins (100 ⁇ g of pooled fractions 6-9).
  • FIG. 5 shows Coomassie Brilliant Blue R250-stained 2D SDS-PAGE of proteins in IMAC-bound fractions stored at ⁇ 20° C. for 3 months.
  • FIG. 6 shows the results of proteolysis of Met(His) 6 -spp24 (residues 24 to 203) by MC3T3-E1 cell extracts.
  • FIG. 7 shows cellular proliferation of A549 human non-small cell lung cancer cells after 48 hours of the indicated treatment with rhBMP-2, spp24, both rhBMP-2 and spp24, or vehicle alone.
  • FIG. 8 shows subcutaneous tumor formation eight weeks after injection of A549 human non-small cell lung cancer cells co-injected with rhBMP-2, spp24, both rhBMP-2 plus spp24 or vehicle alone.
  • FIG. 9 are radiographs illustrating intratibial tumor formation eight weeks after injection of A549 human non-small cell lung cancer cells co-injected with rhBMP-2, spp24, both rhBMP-2 plus spp24 or vehicle alone.
  • FIG. 10 shows representative histological sections of subcutaneous tumors eight weeks after injection of A549 human non-small cell lung cancer cells co-injected with rhBMP-2, spp24, both rhBMP-2 plus spp24 or vehicle alone.
  • FIG. 11 shows histologic analyses of intratibial tumors eight weeks after injection of A549 human non-small cell lung cancer cells.
  • composition comprises a bioactive agent in an effective amount that sequesters:
  • BMP bone morphogenetic protein
  • TGF- ⁇ transforming growth factor-beta family of cytokines
  • composition is effective for a disorder in a mammalian subject.
  • the bioactive agent comprises secreted phosphorprotein 24 kD (spp24) (SEQ ID NO:1) or a fragment thereof, wherein the spp24 is in an effective amount for suppressing or delaying tumor growth.
  • the spp24 is capable of sequestering one of bone morphogenetic protein-2 (BMP-2) and morphogenetic protein-7 (BMP-7) and as least one transforming growth factor-beta (TGF- ⁇ ) family of cytokines so as to suppress or slow tumor growth in a mammalian subject.
  • BMP-2 bone morphogenetic protein-2
  • BMP-7 morphogenetic protein-7
  • TGF- ⁇ transforming growth factor-beta family of cytokines
  • the tumor can be any tumor.
  • the tumor is a metastatic cancer or a primary cancer.
  • the cancer can be metastatic or primary tumor selected from a tumor in bone, a lung tumor, a liver tumor, a brain tumor, a spinal tumor, a breast cancer, or a prostate cancer.
  • the tumor can be any tumor type or individual tumor the growth of which is enhanced by growth factors of the TGF-beta family (e.g., a tumor that manifests with osteoblastic and/or bone metastases).
  • the fragment of spp24 has a molecular weight from about 14 kD to about 20 kD.
  • the spp24 fragment can have amino acids 80-129.
  • the composition can be formulated in a formulation suitable for systemic or local delivery.
  • the formulation is a systemic delivery formulation.
  • the formulation is a local delivery formulation.
  • local delivery formulation include, e.g., formulation for local injection or injection into a tumor, and a sustained release formulation.
  • systemic delivery formulation can be, e.g., formulation for intravenous injection or subcutaneous injection.
  • sustained release formulation can comprise, e.g., an implant or a patch to be administered at a site needing treatment.
  • the composition can be a local delivery formulation comprising a patch suitable for delivery to the site of tumor.
  • composition of the various embodiments above can further comprise a pharmaceutically acceptable carrier.
  • a method of treating or ameliorating a tumor comprising administering to a patient a composition comprising spp24 or a fragment thereof, wherein the spp24 is in an effective amount for suppressing or delaying tumor growth.
  • the spp24 is capable of sequestering one of bone morphogenetic protein-2 (BMP-2) and morphogenetic protein-7 (BMP-7) and as least one transforming growth factor-beta (TGF- ⁇ ) family of cytokines so as to suppress or slow tumor growth in a mammalian subject.
  • the fragment of spp24 has a molecular weight from about 14 kD to about 20 kD.
  • the composition can be formulated in a formulation suitable for systemic or local delivery.
  • the formulation is a systemic delivery formulation.
  • the formulation is a local delivery formulation.
  • local delivery formulation include, e.g., formulation for local injection or injection into a tumor, and a sustained release formulation.
  • systemic delivery formulation can be, e.g., formulation for intravenous injection or subcutaneous injection.
  • sustained release formulation can comprise, e.g., an implant or a patch to be administered at a site needing treatment.
  • the composition can be a local delivery formulation comprising a patch suitable for delivery to the site of tumor.
  • composition of the various embodiments above can further comprise a pharmaceutically acceptable carrier.
  • a method of making a composition comprises forming a composition of the various embodiments described above and below.
  • a method of producing recombinant secreted phosphorprotein 24 kD (spp24) or a fragment thereof comprises expressing a gene comprises a nucleotide sequence encoding spp24 (SPP24) in an expression system.
  • SPP24 comprises a nucleotide sequence of SEQ ID NO:2.
  • the expression system comprises a mammalian cell, a plant cell, yeast, bacteria or is a cell-free expression system.
  • the expression system comprises E. coli.
  • Spp24 is documented to regulate bioactivities of BMP.
  • the present invention discloses that the tumor suppression activities of spp24 are related to its ability to regulate BMP.
  • SPP24 maps to a region of the human genome associated with QTL linked to BUA (Wilson, 2004, Swallow, 1997, Bennett, 2004). Recently-published data confirm that spp24 and its derivatives are a new family of extracellular matrix phosphoproteins that contain a BMP-2-binding TRH1 or pseudoreceptor domain capable of modulating the rate and magnitude of bone formation (Behnam, K., et al., J. Orthop. Res., 23, 175-180 (2005), Sintuu C, et al., J Orthop Res 2008; 26:753-758).
  • SPP24 may be particularly important in early development and the acquisition of peak bone mass. SPP24 was not previously associated with the genetic regulation of bone mass, although
  • Spp24 and its major degradation product can be BMP-binding proteins because they share a common a TGF-beta receptor II homology-1 (TRH1) domain (Behnam, 2005).
  • TRH1 domain TGF-beta receptor II homology-1 domain
  • the synthetic, cyclic N- to C-terminally disulfide bonded peptide corresponding to the 19 amino acid residues of the TRH1 domain of spp18.5 and spp24 specifically binds recombinant human BMP-2 (rhBMP-2) and increases the rate and magnitude of BMP-2-mediated ectopic bone formation in vivo, when assessed histologically or densitometrically (Behnam, 2005).
  • Spp24 is a BMP-2-binding pseudoreceptor that modulates cytokine bioactivity and has significant effects on BMP-2-mediated bone formation in vivo.
  • SPP24 gene for secreted phosphoprotein-24 kDa; alternately known as secreted phosphoprotein-2 or SPP2 maps to chromosome 2q37.1 in the interval 233.64-233.67 Mb of the human genome (Swallow J E, et al., Cytogenet Cell Genet. 1997; 79:142; and Bennett C S, et al., Matrix Biol 2004; 22:641-51).
  • SPP24 genes have been described, all of which are found in vertebrates (protein family [Pfam] PF07448) (Bennett, 2004).
  • the spp24 proteins share 50% to 90% sequence identity, and consist of 3 major domains, including: (1) a short N-terminal secretory peptide, (2) a cystatin-(cysteine protease inhibitor)-like or cathelicidin-(neutrophil antimicrobial peptide precursor)-like domain with two internal disulfide bonds, and (3) a variable arginine-rich C-terminal region (Behnam, 2005, Hu, 1995).
  • the archetypical spp24 is bovine (b)-spp24, a 203 amino acid residue protein with a calculated mass of 23.1 kDa and a theoretical pI of 8.4 (Table 1) (ExPASY ProtParam).
  • a signal peptide (residues 1-23) is cleaved, producing a mature protein of 180 residues (b-spp24, residues 24-203) with a calculated mass of 20.5 kDa and a pI prior to modification of 7.86 (Table 1).
  • Tables 1 and 2 summarize some key information on amino acid sequence of domains of spp24 and related domain information.
  • Fragments of spp24 can be made by recombinant genetic engineering methodology or by a hydrolysis process, e.g., proteolysis. Generally, recombinant fragments of spp24 can be made by expressing a gene encoding a spp24 fragment in an expression system. Expressing spp24 in an E. coli system is described below as an example.
  • the term expression system can be a system cell free system or a system comprising a cell of an organism.
  • Such an organism can be any living organism. Examples of such organisms are plant, yeast, bacteria (e.g., E. coli ), an animal (e.g. a mammal), an insect, etc.
  • Dosages of Spp24 or a fragment thereof can be determined according to methods known in the art based on type of agent, the disease, and other factors such as age and gender. Generally, dosages can vary according to the types of agent (spp24 or a fragment thereof), type of disease or disorder (e.g., a tumor, type of tumor, etc), as well age and gender.
  • the dosage of Spp24 or a fragment thereof for tumor is generally in the milligram quantities, e.g., ranging from about 1 mg to about 500 mg, for topical administration.
  • the doses can be, e.g., 1 mg, or a dose from 10 ⁇ g to about 10 mg.
  • dosages may be continuously given or divided into dosages given per a given timeframe.
  • timeframes include but are not limited to every 1 hour, 2 hour, 4 hour, 6 hour, 8 hour, 12 hour, 24 hour, 48 hour, or 72 hour, or every week, 2 weeks, 4 weeks, or every month, 2 months, 4 months, and so forth.
  • the pharmaceutical composition described herein may be administered to a subject in need of treatment by a variety of routes of administration, including orally and parenterally, (e.g., intravenously, subcutaneously or intramedullary), intranasally, as a suppository or using a “flash” formulation, i.e., allowing the medication to dissolve in the mouth without the need to use water, topically, intradermally, subcutaneously and/or administration via mucosal routes in liquid or solid form.
  • the pharmaceutical composition can be formulated into a variety of dosage forms, e.g., extract, pills, tablets, microparticles, capsules, oral liquid.
  • compositions may also be included as part of the pharmaceutical composition pharmaceutically compatible binding agents, and/or adjuvant materials.
  • the active materials can also be mixed with other active materials including antibiotics, antifungals, other virucidals and immunostimulants which do not impair the desired action and/or supplement the desired action.
  • the composition can be formulated into a formulation for bone, which can include a carrier such as collagen, atelocollagen (collagen treated to remove the immunogenic ends), hydroxyapatite, and a polymer, which is further described below.
  • the formulation can comprise a porous matrix or microspheres made of a polymeric material, which is further described below.
  • the polymer can be, e.g., polylactic acid or polylactide (PLA), or poly(lactic acid-co-glycolic acid), or another bioabsorbable polymer.
  • the mode of administration of the pharmaceutical composition described herein is oral.
  • Oral compositions generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets.
  • the aforesaid compounds may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. Some variation in dosage will necessarily occur, however, depending on the condition of the subject being treated.
  • These preparations should produce a serum concentration of active ingredient of from about 0.01 nM to 1,000,000 nM, e.g., from about 0.2 to 40 ⁇ M.
  • a preferred concentration range is from 0.2 to 20 ⁇ M and most preferably about 1 to 10 ⁇ M.
  • the concentration of active ingredient in the drug composition itself depends on bioavailability of the drug and other factors known to those of skill in the art.
  • the mode of administration of the pharmaceutical compositions described herein is topical or mucosal administration.
  • a specifically preferred mode of mucosal administration is administration via female genital tract.
  • Another preferred mode of mucosal administration is rectal administration.
  • polymeric and/or non-polymeric materials can be used as adjuvants for enhancing mucoadhesiveness of the pharmaceutical composition disclosed herein.
  • the polymeric material suitable as adjuvants can be natural or synthetic polymers.
  • Representative natural polymers include, for example, starch, chitosan, collagen, sugar, gelatin, pectin, alginate, karya gum, methylcellulose, carboxymethylcellulose, methylethylcellulose, and hydroxypropylcellulose.
  • Representative synthetic polymers include, for example, poly(acrylic acid), tragacanth, poly(methyl vinylether-co-maleic anhydride), poly(ethylene oxide), carbopol, poly(vinyl pyrrolidine), poly(ethylene glycol), poly(vinyl alcohol), poly(hydroxyethylmethylacrylate), and polycarbophil.
  • Other bioadhesive materials available in the art of drug formulation can also be used (see, for example, Bioadhesion—Possibilities and Future Trends, Gurny and Junginger, eds., 1990).
  • dosage values also varies with the specific severity of the disease condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted to the individual need and the professional judgment of the person administering or supervising the administration of the aforesaid compositions. It is to be further understood that the concentration ranges set forth herein are exemplary only and they do not limit the scope or practice of the invention.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.
  • the formulation may contain the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, corn starch and the like; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; and a sweetening agent such as sucrose or saccharin or flavoring agent such as peppermint, methyl salicylate, or orange flavoring may be added.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, corn starch and the like
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin or flavoring agent such as pepper
  • dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings.
  • tablets or pills may be coated with sugar, shellac, or other enteric coating agents.
  • Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
  • the solutions or suspensions may also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methylparabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylenediaminetetraacetic acid
  • compositions of the present invention are prepared as formulations with pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carriers Preferred are those carriers that will protect the active compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatable polymers can be used, such as polyanhydrides, polyglycolic acid, collagen, and polylactic acid. Methods for preparation of such formulations can be readily performed by one skilled in the art.
  • Liposomal suspensions are also preferred as pharmaceutically acceptable carriers.
  • Methods for encapsulation or incorporation of compounds into liposomes are described by Cozzani, I.; Joni, G.; Bertoloni, G.; Milanesi, C.; Sicuro, T. Chem. Biol. Interact. 53, 131-143 (1985) and by Jori, G.; Tomio, L.; Reddi, E.; Rossi, E. Br. J. Cancer 48, 307-309 (1983). These may also be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.
  • liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.
  • appropriate lipid(s) such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol
  • the pharmaceutical composition described herein may be administered in single (e.g., once daily) or multiple doses or via constant infusion.
  • the compounds of this invention may also be administered alone or in combination with pharmaceutically acceptable carriers, vehicles or diluents, in either single or multiple doses.
  • Suitable pharmaceutical carriers, vehicles and diluents include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents.
  • the pharmaceutical compositions formed by combining the compounds of this invention and the pharmaceutically acceptable carriers, vehicles or diluents are then readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, injectable solutions and the like.
  • These pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients and the like according to a specific dosage form.
  • tablets containing various excipients such as sodium citrate, calcium carbonate and/or calcium phosphate may be employed along with various disintegrants such as starch, alginic acid and/or certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and/or acacia.
  • various lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tabletting purposes.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules. Preferred materials for this include lactose or milk sugar and high molecular weight polyethylene glycols.
  • the active pharmaceutical agent therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and/or combinations thereof.
  • solutions of the compounds of this invention in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solutions may be employed.
  • aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • the sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art.
  • the compounds of the invention are conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurized container or nebulizer may contain a solution or suspension of a compound of this invention.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound or compounds of the invention and a suitable powder base such as lactose or starch.
  • composition described herein can be formulated alone or together with the other agent in a single dosage form or in a separate dosage form.
  • Methods of preparing various pharmaceutical formulations with a certain amount of active ingredient are known, or will be apparent in light of this disclosure, to those skilled in this art.
  • For examples of methods of preparing pharmaceutical formulations see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition (1995).
  • composition of the various embodiments disclosed above can be formulated into implants, scaffolds, patches, etc.
  • a pharmaceutical composition of the various described embodiments can be administered to a mammal for treating or preventing a tumor.
  • a mammal encompasses all mammalian subjects including human beings and animals.
  • the tumor can be any tumor.
  • the tumor is a metastatic cancer or a primary cancer.
  • the cancer can be metastatic or primary tumors selected from a tumor in bone, a lung tumor, a liver tumor, a brain tumor, a spinal tumor, a breast cancer, a prostate cancer, or any tumor type or individual tumor the growth of which is enhanced by growth factors of the TGF-beta family (e.g, a tumor that manifests with osteoblastic and/or bone metastases).
  • spp24 Secreted phosphoprotein-24 kDa
  • Bovine spp24 is transcribed as a 203 amino acid residue protein that undergoes cleavage of a secretory peptide to form the mature protein (spp24, residues 24 to 203). While not osteogenic itself, spp24 is degraded to a possibly somewhat osteogenic protein, spp18.5, in bone. Both spp18.5 and spp24 contain a cyclic TRH1 (TGF-beta receptor II homology-1) domain similar to that found in the receptor itself and in fetuin.
  • TGF-beta receptor II homology-1 cyclic TRH1 domain similar to that found in the receptor itself and in fetuin.
  • a synthetic peptide including 19 amino acids which is cyclic and derived from the sequence of the bovine protein, called Bone Morphogenetic Protein Binding Peptide or BBP, aka cbBBP (cyclic, bovine BBP), corresponding to the TRH1 domain of spp18.5 and spp24, specifically binds BMP-2 and enhances the rate and magnitude of BMP-2-induced ectopic bone formation in vivo.
  • BBP Bone Morphogenetic Protein Binding Peptide
  • cbBBP cyclic, bovine BBP
  • the parental protein, spp24 exhibits a high affinity for bone and mineral complexes, but its abundance there is low, suggesting that it is rapidly degraded.
  • the availability of recombinant spp24 and its degradation products would facilitate the elucidation of their structure: function relationships.
  • the recombinant spp24 protein was resistant to proteolysis by MC3T3-E1 osteoblastic cell extracts in the absence of calcium; however, in the presence of 4 mM Ca, it can undergo essentially complete proteolysis to small peptides, by-passing the 16 kDa and 14.5 kDa intermediates. This confirms the proteolytic susceptibility of spp24. It also suggests that the levels of spp24 in bone may be regulated, in part, by calcium-dependent proteolysis mediated by osteoblastic cells.
  • the pET20b expression system E. coli BL21(DE3) host strain, media, antibiotics, protein extraction reagents, lysozyme, His-Tag monoclonal antibody, and Western blotting reagents were from Novagen (EMD Biosciences, La Jolla, Calif.). Protein assay kits and electrophoresis supplies, including precast 4% to 20% polyacrylamide gradient minigels, were from Pierce Chemical Company (Rockford, Ill.).
  • His 6 -protease inhibitor (containing [4-(2-aminoethyl)benzenesulfonyl fluoride HCl] or AEBSF, bestatin HCl, [N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide] or E64, pepstatin A, and disodium phosphoramidon), salts and buffers were from Sigma Chemical Company (St. Louis, Mo.). HiTrap IMAC Fast Flow Sepharose 6 columns were from GE Healthcare (Uppsala, Sweden). Affinity-purified rabbit anti-bovine spp24 (residues 168 to 180) was obtained from Genemed (San Francisco, Calif.).
  • SPP24 (SEQ ID NO:2) was commercially cloned from a bovine liver cDNA library by RT-PCR of SPP24-specific primers.
  • the complete nucleotide sequence for the mature, secreted isoform of bovine spp24 (corresponding to amino acid residues 24 to 203) plus an N-terminal Met-(His) 6 extension was cloned into a vector (pcDNA3.1/V5-H isA; Invitrogen, Carlsbad, Calif.). The entire plasmid sequence was confirmed by DNA sequencing (Genedynamics, Portland, Oreg.).
  • the coding sequence was amplified from the plasmid 24 kD/pcDNA3.1/V5-His A using a 5′NdeI-HIS primer and a 3′-HindIII primer, and inserted into the pET20b vector according to the manufacturer's instructions (EMD Biosciences, La Jolla, Calif.).
  • the HIS-SPP24 coding region was sequence-verified on two strands.
  • the plasmid was transformed into E. coli cell line BL21(DE3) and maintained as a bacterial stab culture in 100 ⁇ g/ml ampicillin.
  • Transfected E. coli were seeded onto imMedia Amp Agar (Invitrogen, Carlsbad, Calif.). Single colonies were cultured in 50 ml of imMedia Liquid Amp, grown for 4 to 6 hr at 37° C. with orbital shaking (120 rpm), pelleted by low-speed centrifugation at room temperature, resuspended in 50 ml of fresh imMedia Liquid Amp plus carbenicillin (50 ⁇ g/ml), and cultured for 3 to 4 hr. This inoculum was added to 1 liter of Overnight Express Instant TB Medium plus 1% glycerol. The cells were cultured to stationary phase in a shaking water bath at 37° C. for 16 hr.
  • Inclusion bodies were prepared and solubilized using BugBuster protein extraction reagents as outlined by the manufacturer (Novagen, La Jolla, Calif.) with the addition of His 6 protease inhibitor cocktail (Sigma, St. Louis, Mo.) at all steps. Briefly, the cells were pelleted by centrifugation at 10,000 ⁇ g for 10 min at 4° C. in a Sorval GSA rotor. The supernatant was discarded, and the wet mass of the cell pellets was determined gravimetrically.
  • the wet pellets were resuspended in 5 ml of BugBuster protein extraction reagent and 20 ⁇ l of lysonase per gram of wet pellet weight (initial suspension volume) plus 1% (v/v) His 6 protease inhibitor cocktail.
  • the cell suspension was incubated with shaking (120 rpm) at room temperature for 20 min.
  • the extract was centrifuged at 4° C. for 20 min at 16,000 ⁇ g in a Sorval SS-34 rotor. The supernatant was discarded, and the pellet was resuspended in the same volume of BugBuster protein extraction reagent and protease inhibitor cocktail.
  • a 6-fold volume of 0.1 ⁇ BugBuster and protease inhibitor cocktail was added with vortexing for 1 min.
  • the suspension was centrifuged at 5,000 ⁇ g for 15 min at 4° C. in a Sorval GSA rotor. The supernatant was discarded.
  • the pellet was resuspended in half of the initial suspension volume of 0.1 ⁇ BugBuster and His 6 protease inhibitor cocktail, mixed by vortexing, and centrifuged for 15 min at 5,000 ⁇ g in a Sorval SS-34 rotor at 4° C.
  • the pellet was resuspended in 0.1 ⁇ BugBuster plus protease inhibitor cocktail, vortexed, and centrifuged for 15 min at 10,000 ⁇ g in a Sorval SS-34 rotor at 4° C. The supernatant was discarded, and the pellet containing the inclusion bodies was subjected to IMAC, as described in detail below.
  • His 6 -tagged spp24 was isolated from inclusion bodies by immobilized metal affinity chromatography (IMAC) on HiTrap IMAC FF columns (GE Healthcare Amersham Biosciences, Uppsala, Sweden) using a BioLogic protein purification workstation (BioRad, Hercules, Calif.).
  • IMAC immobilized metal affinity chromatography
  • HiTrap IMAC FF columns GE Healthcare Amersham Biosciences, Uppsala, Sweden
  • BioLogic protein purification workstation BioRad, Hercules, Calif.
  • the columns Prior to use, the columns were washed with 10 ml of distilled water to remove residual ethanol, charged with 0.5 ml of 100 mM cobalt chloride in distilled water, washed with 5 ml of distilled water, and pre-equilibrated with 10 ml of filtered, degassed Buffer A (8 M deionized urea, 100 mM sodium phosphate buffer [pH 7.4], 20 mM imidazole).
  • the inclusion body pellet derived from 1 liter of cells was suspended in 5 ml of buffer A, transferred to 1.7-ml microcentrifuge tubes, centrifuged at maximum speed (14,000 rpm) in a Beckman model 5415C microfuge for 2 min to pellet insoluble material, then applied to a loading loop.
  • the fraction size was 1.0 ml.
  • the column was pre-equilibrated with 5 ml of buffer A to achieve a stable baseline, and the sample was loaded onto the column through a 1.0-ml static loop. Multiple injections were required to load sample volumes >1.0 ml.
  • the unbound proteins were isocratically eluted with 10 ml of Buffer A.
  • Bound proteins were eluted with a 10 ml linear gradient of 0% to 100% buffer B (8 M urea, 100 mM sodium phosphate [pH 7.4], 500 mM imidazole), followed by 10 ml of 100% buffer B. A 3-ml linear gradient from 100% to 0% buffer B, and 10 ml of isocratic flow at 100% buffer A were used to return the column to baseline conditions.
  • the proteins in representative 1-ml IMAC fractions containing His 6 -spp24 and its degradation products were passed through a C18 reverse phase tip to desalt them, then subjected to high-resolution MS to determine their precise molecular weights on a fee-for-service basis (Mary Ann Gawinowicz, Ph.D., Protein Chemistry Core Facility, Howard Hughes Medical Institute, Columbia University, New York, N.Y.).
  • Visualization of His 6 -tagged spp24 was accomplished by Western blotting using a primary antibody and colorimetric kit from Novagen (EMD Biosciences, La Jolla, Calif.). Visualization of the C-terminal domain of bovine spp24 (residues 168 to 180) was accomplished by Western blotting using an affinity-purified rabbit antibody to the keyhole limpet-conjugated peptide and a kit from Vector Laboratories (Burlingame, Calif.). The primary antibodies (mouse monoclonal anti-His.Tag and rabbit anti-bovine spp24 [residues 168 to 180]) were used at a final concentration of 1 ⁇ g/ml.
  • the secondary antibody directed against the mouse monoclonal anti-His.Tag was goat anti-mouse IgG conjugated to alkaline phosphatase, and it was used at a 1:5000 dilution.
  • the secondary antibody directed against the rabbit anti-bovine spp24 was donkey anti-rabbit IgG conjugated to alkaline phosphatase, and it was used at a 1:2500 dilution. Color development was accomplished with BCIP/NBT in alkaline buffer.
  • the proteins in representative fractions containing His 6 -spp24 and its degradation products were separated by 2D SDS-PAGE by the method of O'Farrell (O'Farrell P. H., J. Biol. Chem., 250, 4007-4021 (1975)) (Nancy Kendrick, Ph.D., Kendrick Laboratories, Madison, Wis.) (Behnam, 2005, Behnam, 2006; Behnam, 2002; and Behnam and Murray, 2005). These fractions had been stored at ⁇ 20° C. for 3 months in urea buffer plus protease inhibitors, resulting in the slow accumulation of degradation products similar to those previously reported (Urist, 1987) that are not abundant in freshly-prepared fractions.
  • the gel was stained with Coomassie Brilliant Blue, and the three major spots of the appropriate molecular weight ( ⁇ 24 kDa) were selected, excised, eluted, reduced in 5% ⁇ -mercaptoethanol, alkylated, trypsin digested, and subjected to peptide mass fingerprint analysis by MALDI/ToF MS (Mary Ann Gawinowicz, Ph.D., Protein Chemistry Core Facility, Howard Hughes Medical Institute, Columbia University, New York, N.Y.) to verify their identities and structures (Behnam, 2005, Behnam, 2006, Behnam, 2002; and Behnam and Murray, 2005). Methionines were not oxidized. Peptides with a mass [M+H + ]>1000 Da were analyzed.
  • the peptide fingerprint was compared to those in the SWISS-PRO and NCBI databanks (Behnam, 2005, Behnam, 2006; Behnam, 2002; and Behnam and Murray, 2005).
  • the spots in replicate gels were subjected to N-terminal sequencing by automated Edman degradation. 2D gels were used for these analyses because the resolution of closely-related proteins is greater, permitting easier identification, excision, and post-electrophoresis characterization, such as N-terminal sequencing.
  • MC3T3-E1 mouse preosteoblastic cells were cultured to confluence, harvested by trypsinization, pelleted by centrifugation, and suspended in digestion buffer (100 mM HEPES-HCl, pH 7.5, 12.5 mM KCl, 6.25 mM (3-mercaptoethanol, and 0.2% Triton X-100) plus or minus 4 mM CaCl 2 at a protein concentration of 2.4 mg/ml.
  • Recombinant spp24 (residues 23 to 204) was exhaustively dialyzed vs. water, lyophilized, and resuspended at 1 mg/ml in digestion buffer (plus or minus 4 mM CaCl 2 ).
  • Equal volumes of cell extract and spp24 were combined, and incubated at 37° C. for 0 to 24 hours. At the intervals indicated, 4 ⁇ l aliquots equivalent to 2 ⁇ g spp24 were removed, subjected to SDS-PAGE, and Western blotted using antibody directed against the His 6 tag as outlined in detail above.
  • FIG. 1 The map of the HIS-SPP24 pET20B expression vector is shown in FIG. 1 .
  • DNA sequencing confirmed that it corresponds to a construct with an N-terminal Met followed by a His 6 -tag, and residues 24 through 203 of bovine spp24 ( FIG. 2 ).
  • FIG. 2 When plasmid expression was induced in the host E. coli strain [BL21(DE3)], the recombinant protein appeared in significant amounts in the inclusion bodies, as illustrated by the cobalt-binding peak in fractions 19 through 24 in the chromatogram ( FIG. 3 ). Unbound proteins eluted in Fractions 6 to 8.
  • the apparent doublet represents flow through of unbound proteins following two injections of solubilized inclusion body proteins through a 1.0-ml static injection loop, with a brief pause between injections.
  • the His 6 -tagged spp24 proteins eluted as a single peak starting at 19% Buffer B (100 mM imidazole). The yield was 5 to 8 mg His 6 -spp24 per liter of cultured bacteria.
  • Freshly IMAC-purified fractions of spp24 contained a single major protein band at about 21 kDa on 1D SDS 4% to 20% polyacrylamide gradient gels ( FIG. 4 , Lane 3) that was confirmed to be His 6 -spp24 by Western blotting ( FIG. 4 , Lane 4).
  • the 21 kDa protein is highly susceptible to proteolysis, even when stored frozen in 8 M urea in the presence of protease inhibitors.
  • 2D SDS-PAGE gels of IMAC-purified His 6 -spp24 that had been frozen at ⁇ 20° C. for 3 months showed a much more complex pattern of proteins ( FIG. 5 ) than the one that was observed on 1D gels of freshly-purified protein ( FIG. 4 ).
  • 2D SDS-PAGE resolved at least 3 major groups of proteins that ranged in apparent mass from about 21 kDa (Spot 1) to about 14 kDa (a major doublet including Spot 3), with a series of overlapping protein spots of intermediate mass of around 16 kDa (includes Spot 2) ( FIG.
  • FIG. 3 shows the chromatogram showing fractionation of unbound inclusion body protein (Fractions 6-9) and His 6 -tagged spp24 (Fractions 19-23) by FF IMAC chromatography.
  • the IMAC column was initially charged with cobalt.
  • Blue line UV absorbance (AU); Red line: Conductivity (mS/cm); Black line: % B.
  • Buffer A 8 M urea, 100 mM sodium phosphate buffer [pH 7.4], 20 mM imidazole; Buffer B: Buffer A+100 mM imidazole.
  • FIG. 4 shows Coomassie-blue stained 4% to 20% polyacrylamide gradient gels and Western blots showing proteins in freshly-prepared IMAC column fractions.
  • Lane 1 Molecular weight standards.
  • Lane 2 Unbound proteins (100 ⁇ g of pooled fractions 6-9).
  • Lane 3 5 ⁇ g of dialyzed spp24 (pooled bound fractions).
  • Lane 4 Western blot of proteins in Lane 3 showing immunopositive His 6 -spp24 bands in IMAC fractions. Note that a single major band at 21 kDa is observed in this freshly-prepared preparation of spp-24.
  • Freshly-isolated His 6 -spp24 was incubated with extracts of confluent mouse MC3T3-E1 osteoblastic cells in the absence and presence of 4 mM total calcium. In the absence of added calcium, essentially no degradation was observed until 24 hr of incubation ( FIG. 6 ). However, in the presence of calcium, essentially complete degradation of His 6 -spp24 was observed in about 2 hours ( FIG. 6 ), confirming the susceptibility of the protein to proteolysis. The proteolysis observed in the presence of calcium differs from the proteolysis observed during long-term storage of His 6 -spp24. In the presence of calcium, spp24 is rapidly degraded to small peptides ( ⁇ 7 kDa) ( FIG. 6 ), while long-term storage in the absence of calcium is associated with the gradual appearance of two proteins (spp14.5 and spp16) of intermediate size ( FIG. 5 ).
  • FIG. 6 shows the results of proteolysis of Met(His) 6 -spp24 (residues 24 to 203) by MC3T3-E1 cell extracts.
  • the recombinant protein was incubated with solubilized cell extracts in the absence or presence of 4 mM calcium for up to 24 hr. Aliquots corresponding to 2 ⁇ g of spp24 were removed, subjected to SDS-PAGE, and Western blotted with anti-His 6 as outlined in the text. In the presence of calcium, essentially all of the spp24 is degraded within about 2 hours. No accumulation of proteins with intermediate molecular weights of 14.5 kDa and 16 kD is observed in the presence of calcium.
  • Secreted phosphoprotein-24 kDa is a relatively rare extracellular matrix protein (Hu, 1995, Behnam, 2005, Urist, 1987). The highest levels of the intact 24 kDa protein and its major degradation product (spp18.5) are observed in bone mineral (Hu, 1995, Urist, 1987). Although trace amounts of spp24 are also present in fetuin-mineral complexes (Price, 2003), and spp24 transcripts are expressed in a variety of other vertebrate mesodermal tissues (Okazaki, 2002; Strausberg, 2002; and Bennett, 2004), its functions remain poorly characterized.
  • Carbamoylation of “BMP/NCP” by cyanate ions may account for the N-terminal blockage of the native protein isolated from bone, as well as the complex MS profiles observed in recombinant Met(His) 6 -spp24 (residues 24-203) and its 16 kDa and 14.5 kDa degradation products (Table 3).
  • the 8 M urea used in the buffers described was deionized prior to use to remove cyanate ions, but this did not prevent carbamoylation.
  • Spp24 contains two major functional domains, including a cystatin (cysteine-protease inhibitor) domain and a TRH-1 domain within the larger cystatin domain, which may impart significant, but different, roles in regulating bone formation, turnover, and repair to the secreted protein and its degradation products.
  • cystatin cystatin-protease inhibitor
  • TRH-1 domain within the larger cystatin domain
  • the most common degradation product was the 16 kDa protein [Met(His) 6 -spp24, residues 23 to 157], while a less abundant 14.5 kDa degradation product [Met(His) 6 -spp24, residues 23 to 143] was also observed. These cleavages occur between residues 157 and 158 and residues 143 and 144 of native bovine spp-241-203, respectively (Table 4).
  • Calpains calcium-activated papain-like proteases are abundant proteins in osteoblasts (Tram, K.K.-T., et al., Biochem. Mol. Biol. Intl., 29, 981-987 (1993)) and may be one of the proteolytic enzyme systems that mediate spp24 degradation in the presence of MC3T3-E1 cell extracts and calcium. Experiments are currently underway to identify and characterize the protease(s) that contribute to the proteolytic processing of spp24.
  • SVAKVNSQSL 50 (SEQ ID NO: 15) (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18) SPYLFRAFRS 60 SVKRVNALDE 70 DSLTMDLEFR 80 IQETTCRRES 90 (SEQ ID NO: 19) (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) EADPATCDFQ 100 RGYHVPVAVC 110 RSTVRMSAEQ 120 VQNVWVRCHW 130 (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 25) (SEQ ID NO: 26) SSSSGSSSSE 140 EMF ⁇ FGDILGS 150 STSRNSY ⁇ LLG 160 LTPDRSRGEP 170 (SEQ ID NO: 27) (SEQ ID NO: 28) (SEQ ID NO: 15) (SEQ ID NO: 29) LYEPSREMRR 180 NFPLGNR
  • Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor-beta (TGF- ⁇ ) family of proteins. These proteins perform a variety of pattern specifying and morphogenic functions in the mammalian embryo and are critical to skeletal homeostasis in post-natal life.
  • Recombinant human BMP-2 (rhBMP-2) is well accepted as an osteoinductive therapeutic which is used to promote bone healing in fracture non-unions and spine fusion in degenerative disc diseases.
  • BMPs can be significant in pathological processes such as skeletal metastasis.
  • Secreted phosphoprotein 24 kD is a bone matrix protein which binds proteins of the TGF- ⁇ family through a region that is similar to the TGF- ⁇ receptor II (TRH1 domain) (Behnam, 2005).
  • Full-length spp24 strongly inhibits BMP-2 induced bone formation when tested in an ectopic bone forming assay (Sintuu, 2008).
  • Smaller forms of spp24 produced through proteolysis are less inhibitory of BMP-2 induced bone formation (Brochmann E J, et al., J Orthop Res 2010; 28:1200-7) which has lead to the hypothesis that regulated proteolysis of spp24 is one mechanism through which the availability of BMPs is regulated in skeletal tissue (Brochmann, 2009).
  • Cell line A549 is a well characterized human non-small cell lung carcinoma line that expresses BMP-2 and responds to this autocrine secretion with increased growth. Previous investigations have shown that noggin, an inhibitor of BMPs, can be employed to reduce tumor growth in this model of skeletal metastasis (Feeley B T, et al., J Bone Min Res 2006; 21:1571-1580).
  • BMPs and TGF- ⁇ contribute to the growth of some skeletal metastases through autocrine stimulation.
  • Spp24 has been shown to bind to both BMP-2 and TGF- ⁇ and to markedly inhibit the osteogenic properties of rhBMP-2.
  • the studies in this example would show that addition of spp24 would sequester autocrine growth factors (especially BMP-2) and reduce tumor growth in a system where autocrine stimulation by BMP-2 is known to be important.
  • spp24 could be used to bind to and sequester BMP-2 (and other related cytokines) and through this interruption of autocrine stimulation decrease tumor growth.
  • the compositions of invention therefore would have applications in the orthopaedic management of metastatic disease.
  • the human non-small cell lung cancer (NSCLC) cell line A549 was used in this study (ATCC, Manassas, Va., USA).
  • A549 cells were maintained in DMEM with 10% FBS and antibiotics (Fisher scientific, Pittsburgh, Pa.) in a humidified incubator with 5% CO2 at 37° C. Cells were not used beyond 10 passages. Cells were plated in serum free medium at a density of 10,000 cells/well and allowed to attach for 24 h.
  • RhBMP-2 (INFUSE®, Medtronic Sofamor Danek, Minneapolis, Minn.) was added to fresh serum-free media at final concentrations of 0, 1, 10, 50, 100, and 500 ng/ml.
  • Recombinant spp24 was prepared as described previously (Murray E J B, et al., Connect Tiss Res 2007; 48:1-8) and added in serum-free media to final concentrations of 10, 25, 50, 75 ⁇ g/ml. Cells were incubated for 48 h before cell proliferation was assessed using Quick Cell Proliferation Assay (BioVision, Mountain View, Calif., USA) according to manufacturer's recommendations. Results were reported as a percentage compared with untreated cells.
  • mice Forty, eight-week-old male SCID mice (weight 23.5 to 29.1 g, mean 27.1 g) were housed under pathogen-free conditions in accordance with the protocol approved by the Chancellor's Animal Research Committee (ARC) at the University of California, Los Angeles. There were four experimental groups in this study, and each group underwent implantation of cells in both a subcutaneous model and an intra-tibial injection. Group I animals received A549 cells alone as a control group. Group II animals received A549 cells+rhBMP-2 (10 ⁇ g/10 ⁇ l). Group III received an A549 cells+spp24 (1000 ⁇ g). Group IV received an A549 cells+rhBMP2+spp24. There were 10 animals in each group.
  • A549 cells (1 ⁇ 10 5 ) were suspended in 15 ⁇ l of 1 ⁇ PBS and 15 ⁇ l Affi-Gel Blue (BioRad Laboratories, Hercules, Calif.) and injected into the subcutaneous space on the backs of mice. Briefly, after the SCID mice were anesthetized, then maintained via an isoflurane face mask, the overlying skin was prepped in sterile fashion with 70% ethanol and betadine. Different materials and A549 cells were then injected into the subcutaneous space on the backs of SCID mice. Animals were killed at 8 weeks or earlier if large tumor size exceeded that permitted by the ARC protocol and the tumor removed. Tumors were measured in three dimensions (length ⁇ width ⁇ depth). These measurements were repeated by three observers blinded to treatment group. Results are reported in millimeters as mean ⁇ SE.
  • Tibial implantation of A549 cells plus experimental materials was performed as previously described. 14,15 Mice were anesthetized with isoflurane, then maintained via an isoflurane face mask. The overlying skin was prepped in a sterile fashion with 70% ethanol and betadine. A 3-mm longitudinal incision was made over the patellar ligament with a no. 11 scalpel blade and a 2-mm longitudinal incision was then made along the medial border of the patellar ligament to the tibial plateau. A 27.5-gauge needle was introduced through the proximal tibial plateau and into the proximal tibia.
  • BMP-2 significantly increased proliferation in a dose-dependent manner at doses of the 0, 1, 10, and 50 ng/ml. Higher doses (50 and 500 ng/ml) were associated with a decline in proliferation rates towards those of the cells alone group.
  • the addition of spp24 to the BMP treatment resulted in an attenuation of the BMP affect at all doses. Treatment of the cells with spp24 alone resulted in a slight dose-dependent inhibition of proliferation.
  • FIG. 8 The results of measurements of tumor size following subcutaneous injection of A549 cells with the various treatments are shown in FIG. 8 .
  • Addition of rhBMP-2 increased tumor volume by about 3 fold.
  • addition of spp24 dramatically reduced tumor volume in both the group that was treatment with exogenous BMP and the group where cells were delivered with only spp24.
  • FIG. 8 shows subcutaneous tumor formation eight weeks after injection of A549 human non-small cell lung cancer cells co-injected with rhBMP-2, spp24, both rhBMP-2 plus spp24 or vehicle alone.
  • Images illustrate representative tumors at necropsy. For numerical analysis of measurements from all tumors
  • Radiographs of injected tibias were obtained at the time of death and evaluated by three independent observers who were blinded with respect to treatment groups. There was excellent overall agreement between readers with a kappa statistic of 0.89. These results are presented in FIG. 9 .
  • FIG. 9 shows intratibial tumor formation eight weeks after eight weeks after injection of A549 human non-small cell lung cancer cells co-injected with rhBMP-2, spp24, both rhBMP-2 plus spp24 or vehicle alone. Comparisons with significant differences (p ⁇ 0.05) are indicated with an asterisk. Radiographs illustrate representative findings at the time of necropsy.
  • Specimens from the cells alone and the cells plus rhBMP-2 treatment groups showed abundant masses of adenocarcinoma cells in both the subcutaneous ( FIG. 10 ) and intratibial ( FIG. 11 ) sites whereas those from the Cell+SPP and the Cell+BMP+SPP groups revealed only a few scattered tumor cells.
  • Table 6 shows the calculated results for the % TuV/TV (the proportion of tumor volume to total tissue volume in a longitudinal section of the proximal tibia at midline) and the % By/Tv (the proportion of total mineralized trabeculae volume in the total tissue volume in the section) from the specimens in this study.
  • Animals that were injected with A549 cells plus rhBMP2 had the highest tumor volume but this value was not significantly greater than that of the A549 cells alone group. Both of these groups had higher relatively tumor volumes than those of either group that received spp24 but there was insufficient tumor mass in the specimens from the groups that received spp24 to allow for quantitative analysis.
  • FIG. 10 shows representative histological sections of subcutaneous tumors eight weeks after injection of A549 human non-small cell lung cancer cells co-injected with rhBMP-2, spp24, both rhBMP-2 plus spp24 or vehicle alone.
  • FIG. 11 shows histologic analyses of intratibial tumors eight weeks after injection of A549 human non-small cell lung cancer cells. Animals treated with A549 cells alone and A549 cells+rhBMP-2 showed significant tumor formation in the proximal tibia.
  • Insert in upper left shows high power view of the knee joint
  • B New corticoendosteal bone formation in association with occupation of the intramedullary canal by infiltrating tumor cells (asterisk);
  • C New subperiosteal bone formation (arrows) associated with tumor invasion of muscular tissue and the medullary canal (asterisks);
  • D New (woven) bone formation (arrows) within the tumor mass (asterisks);
  • E Hemorrhage and necrotic areas within a large tumor mass;
  • F Peritumoral angiogenesis (arrows) seen between normal muscle fibers (asterisk) and tumor cells.
  • the vertebral column is the most common site of skeletal metastases (Harrington, I. D: et al., J Bone Joint Surg Am 1986; 68:1110-1115). Metastases from primary tumors of the lungs, prostate, breasts, kidneys, thyroid, and gastrointestinal tract account for the majority of spinal column tumors. Improvements in oncological treatment including adjuvant therapies and more aggressive resection have increased survival time in patients with spinal tumors. Although many tumors are not curative, resection and stabilization can have beneficial effects on neurological status, function, pain, and mobility (see, e.g., Boden, S D, Schimandle J H. Fusion. Biology of lumbar spine fusion and bone graft materials. In: International Society for Study of the Lumbar Spine Editorial Committee, editors. The lumbar spine, 2 nd ed. Philadelphia: WB Saunders, 1996:1284-306).
  • BMPs are expressed in a variety of carcinoma cell lines and tumors originating from multiple organs including tumors of lung (e.g., Langenfeld, E M, et al., Carcinogenesis 2003; 24:1445-1454) prostate (see, e.g., Masuda, H, et al., Prostate 2003; 54:268-74) breast (see, e.g., Pouliot F, et al., J Endocrinol 2000; 172:187-198) and kidney (Kim, I Y, et al., Clin Cancer Res 2003; 9(16 Pt 1):6046-6051).
  • Osteoblastic tumor cells express mRNA and protein for BMP receptors, and exogenous BMPs stimulate tumor cell cellular migration and invasion in vitro and in vivo (Feeley, B T, et al., J Bone Miner Res. 2005; 20:2189-2199). These results suggest, therefore, that BMP/TGF-f3 cytokines are important in the development of metastatic osteoblastic lesions and that inhibition of their activity would effectively limit tumor growth.
  • Secreted phosphoprotein 24 kD is a bone matrix protein which binds proteins of the TGF- ⁇ family through a region that is similar to the TGF- ⁇ receptor II (TRH1 domain) (Behnam, 2005) and inhibits the osteogenic activity of BMP-2 (see, e.g., Brochmann, 2009). This protein exits in bone tissue in several proteolytic size forms which effect BMP-2 activity differently (Brochmann, 2009).
  • spp24 shares sequence and physico-chemical properties with pro-osteogenic proteins described decades ago by two investigators (Urist and Sen) (Brochmann, 2009) and a BMP enhancing therapeutic peptide has been developed which is based on the sequence of the BMP/TGF-beta binding region of spp24 (Behnam, 2005).
  • spp24 had an effect that was similar to that of noggin (e.g., Feeley, 2006).
  • the current therapies for metastatic cancer in bone include hormone treatment (which is ineffective against androgen- or estrogen-independent tumors), external beam radiation (with corticosteroids or surgical decompression of spinal metastases), radioisotopes, prophylactic surgery for osteolytic lesions with impending fracture, bisphosphonates or RANK/RANKL system inhibitors (e.g. osteoprotegerin) for osteolytic bone lesions, and growth factor, growth factor receptor or cell adhesion protein antagonists (Canalis, E, et al., Endo Rev 2003; 24:218-235).
  • the limitations of these approaches include toxicity, specificity (osteolytic vs. osteoblastic lesions), cost, and the potential for adverse side effects (Canalis, 2003).
  • Spp24 can reduce A549 cell tumor growth in both soft tissue and intraosseus environments. We believe, but are not bound by, that the mechanism for this inhibition is interruption of autocrine stimulation through the sequestration of BMP-2. Spp24 can be developed into a therapeutic agent that can be employed in clinical situations where the inhibitions of BMPs and related proteins are advantageous.

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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1996021006A1 (fr) 1995-01-06 1996-07-11 The Regents Of The University Of California Nouvelle phosphoproteine secretee par la matrice extracellulaire d'organes de mammiferes et ses procedes d'utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996021006A1 (fr) 1995-01-06 1996-07-11 The Regents Of The University Of California Nouvelle phosphoproteine secretee par la matrice extracellulaire d'organes de mammiferes et ses procedes d'utilisation

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
Brochmann et al., "Carboxy terminus of secreted phosphoprotein-24 kDa (spp24) is essential for full inhibition of BMP-2 activity", J. of Orthopedic Res. Epub. vol. 28, No. 9, pp. 1200-1207 (2010).
Burris III et al. (1992, Journal of the National Cancer Institute 84:1816-1820). *
Depenbrock et al. (1997, European Journal of Cancer 33:2404-2410). *
Dermer (Bio/Technology, 1994, 12:320). *
Freeley et al., "Mixed Metastatic Lung Cancer Lesions in Bone are Inhibited by Noggin Overexpression and Rank: Fc Administration", J. of Bone and Mineral Res. vol. 21, issue 10, pp. 1571-1580 (2006).
Freshney (Culture of Animal Cells, A Manual of Basic Technique, Alan R. Liss, Inc., 1983, New York, p. 4). *
Gabrielson et al. (1999, Clinical Cancer Research 5:1638-1641). *
Georgoulias (2002, Current Medicinal Chemistry 9:869-877). *
Gura (Science, v278, 1997, pp. 1041-1042). *
Hanauske et al. (1995, Investigational New Drugs 13: 43-49). *
International Search Report for appl. PCT/US2011/037952, mailed Mar. 2, 2012, 4 pgs.
Izbicka et al. (1999, investigational New Drugs 16:221-225). *
Kornblith et al. (2003, Anticancer Research 23:543-548). *
Lawrence et al. (1999, Anti-Cancer Drugs 10:655-661). *
Lee et al., "Effects of the bone morphogenetic protein binding protein spp24 (secreted phosphoprotein 24KD) on the growth of human lung cancer cells", J. of Orthopedic Res. Epub, vol. 29, No. 11 pp. 1712-1718 (2011).
Martin et al. (1994, Journal of the National Cancer Institute 86:608-613). *

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